Baculovirus Expression of Human P450 2E1 and Cytochrome b5: Spectral and Catalytic Properties and Effect of b5 on the Stoichiometry of P450 2E1-Catalyzed Reactions

Abstract

The baculovirus (BV)-insect cell expression system has been successfully used to express high levels of mammalian proteins. Here we report the baculovirus-mediated expression of human P450 2E1 and cytochrome b5 in Sf9 insect cells. The BglI-EcoRI fragment of the human P450 2E1 cDNA (Umeno et al., Biochemistry 27, 1988) was used for the expression of P450 2E1. Human cytochrome b5 cDNA was obtained by the polymerase chain reaction using human liver cDNA as template. Infection of Sf9 insect cells with a recombinant 2E1-BV resulted in the expression of a protein which comigrated with human liver P450 2E1 on SDS gels and cross-reacted with a polyclonal antibody against rat liver P450 2E1. Inclusion of hemin in the cell growth medium greatly enhanced the spectral activity of expressed 2E1. Expression levels of 1.5 nmol/mg cell lysate were obtained after 7 days of infection. However, maximal turnover numbers (min−1) were observed after 48 h of infection. The λmax of the CO:reduced CO difference spectrum of human P450 2E1 was found to be 451.1 nm. In the presence of exogenous hemin, BV-expressed human b5 showed a typical reduced - oxidized difference spectrum with λmax and λmin of 425 and 409 nm, respectively. With saturating levels of purified rat liver NADPH:P450 oxidoreductase and rat liver cytochrome b5 included in the incubations, expressed human P450 2E1 showed high catalytic activity for the metabolism of p-nitrophenol (PNP), ethoxycoumarin, N-nitrosodiethylamine, and N-nitrosodimethylamine (NDMA). The presence of b5; in the incubations increased the activities several-fold. The Km and kcat values for the N-demethylation of NDMA to HCHO in the presence of rat b5 by expressed 2E1 were 36.0 μM and 8.3 min−1, respectively. In the absence of extra added b5, the Km was increased sixfold and the kcat decreased fourfold. In the presence of extra added b5 expressed 2E1 showed a Km for PNP metabolism of 86 μM and a kcat of 7.8 min−1. Simultaneous infection of Sf9 insect cells with both 2E1 and human b5 recombinant BV resulted in a membrane fraction (2E1:b5) containing both proteins at a ratio of b5 to P450 of approximately 1.8:1. The Km and kcat values for NDMA demethylase activity by the 2E1:b5 membrane fraction were similar to those with exogenously added b5. Stoichiometric analysis of the rates of NADPH oxidation and product and H2O2 formation showed that the sum of the HCHO (product) and H2O2 formed could not account for the amount of NADPH utilized which is consistent with a pathway leading to H2O formation. The presence of substrate did not significantly affect the rates of either NADPH oxidation or H2O2 formation. Extra added b5 significantly decreased the rate of NADPH utilization and H2O2 formation by approximately 63 and 86%, respectively, with or without substrate in the incubations. The results show that exogenous b5 decreased H2O2 and H2O formation by an amount which could account for the extra reducing equivalents needed for the increased rate of product formation, suggesting a mechanism for the b5-dependent stimulation.